Pyridyl derivatives as cftr modulators

ABSTRACT

The present invention relates to modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator (“CFTR”), compositions thereof, and methods therewith. The present invention also relates to methods of treating ABC transporter mediated diseases using such modulators.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119 toU.S. provisional patent application Ser. No. 61/040,776, filed Mar. 31,2008, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including Cystic FibrosisTransmembrane Conductance Regulator (“CFTR”), compositions thereof, andmethods therewith. The present invention also relates to methods oftreating ABC transporter mediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a family of membrane transporter proteins thatregulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. ABCtransporters are homologous membrane proteins that bind and use cellularadenosine triphosphate (ATP) for their specific activities. Some ofthese transporters were discovered as multidrug resistance proteins(like the MDR1-P glycoprotein, or the multidrug resistance protein,MRP1), defending malignant cancer cells against chemotherapeutic agents.To date, 48 ABC Transporters have been identified and grouped into 7families based on their sequence identity and function.

ABC transporters regulate a variety of important physiological roleswithin the body and provide defense against harmful environmentalcompounds. Because of this, they represent important potential drugtargets for the treatment of diseases associated with defects in thetransporter, prevention of drug transport out of the target cell, andintervention in other diseases in which modulation of ABC transporteractivity may be beneficial.

One member of the ABC transporter family commonly associated withdisease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressedin a variety of cells types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeate of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced, (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in CysticFibrosis (“CF”), the most common fatal genetic disease in humans. CysticFibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the endoplasmic reticulum (“ER”), and traffic to theplasma membrane. As a result, the number of channels present in themembrane is far less than observed in cells expressing wild-type CFTR.In addition to impaired trafficking, the mutation results in defectivechannel gating. Together, the reduced number of channels in the membraneand the defective gating lead to reduced anion transport acrossepithelia leading to defective ion and fluid transport. (Quinton, P. M.(1990), FASEB J. 4: 2709-2727). Studies have shown, however, that thereduced numbers of ΔF508-CFTR in the membrane are functional, albeitless than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354:526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell.Biochem. 270: 12347-50). In addition to ΔF508-CFTR, other diseasecausing mutations in CFTR that result in defective trafficking,synthesis, and/or channel gating could be up- or down-regulated to alteranion secretion and modify disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺-K⁺-ATPase pumpand Cl− channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to Cystic Fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery, has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),hereditary emphysema (due to a1-antitrypsin; non Piz variants),hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). Sixteen million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrheal causing bacteria is enterotoxogenic E-coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity, particularly CFTR activity.These compounds have the general formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein R^(N),        ring A, ring B, W, R^(w), R¹, n, w, and x are described below.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, diabetesmellitus, laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type I, hereditaryemphysema, congenital hyperthyroidism, osteogenesis imperfecta,hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (di),neurophyseal di, neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, spinocerebullar ataxia typeI, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, andmyotonic dystrophy, as well as spongiform encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjögren'sdisease.

DETAILED DESCRIPTION OF THE INVENTION General Description of Compoundsof the Invention

The present invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R^(N) is H, alkyl, aryl, heteroaryl, aralkyl, cycloalkyl, orheterocycloalkyl;

A is an optionally substituted 3-7 membered monocyclic ring;

B is optionally fused to a 5-7 membered ring selected from the groupconsisting of cycloaliphatic, aryl, heterocyclic, and heteroaryl;

R¹ is halo, alkyl, OH, alkoxy, thioalkyl, trifluoromethoxy; or

two R¹ on adjacent atoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;

W is independently a bond or an optionally substituted (C1-C6)alkylidene chain wherein up to two methylene units of W areindependently replaced by —CO—, —O—, —S—, —SO₂—, or —NR′—;

R¹ is independently H, alkyl, aryl, heteroaryl, aralkyl, cycloalkyl, orheterocycloalkyl;

R^(W) is independently H, halo, CN, NO₂, N(R)₂, CF₃, OCF₃, OH, OR,C(O)N(R)₂, C(O)R, CO₂R, —O(C1-C6)alkylidene-OR,—O(C1-C6)alkylidene-N(R)₂, —O(C1-C6)alkylidene-heterocycloalkyl, or anoptionally substituted aliphatic, cycloaliphatic, aryl, aryloxy,heterocyclic, or heteroaryl, wherein, when substituted, R^(w) issubstituted with up to two R²;

R² is halo, CN, NO₂, CF₃, OCF₃, OR, OC(O)R, OC(O)N(R)₂, SR, S(O)R, SO₂R,SO₂N(R)₂, SO₃R, C(O)R, CO₂R, C(O)N(R)₂, N(R)₂, NRC(O)R, NRCO₂R,NRC(O)N(R)₂, NRSO₂R, B(OH)₂, or NRSO₂N(R)₂;

R is independently H, alkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl;

n is 1 or 2;

w is an integer from 0 to 4 inclusive; and

x is an integer from 0 to 5 inclusive; provided that when W is a bondand R^(w) is cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl,—W—R^(w) is attached to the 3- or 4-position of the pyridyl ring.

COMPOUNDS AND DEFINITIONS

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group” or “alkyl”, as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroaliphatic”, as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, phosphorus, or silicon.Heteroaliphatic groups may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and include “heterocycle”,“heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic” groups.

The term “heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or“heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic,or tricyclic ring systems in which one or a plurality of ring members isan independently selected heteroatom. In some embodiments, the“heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic”group has three to fourteen ring members in which one or more ringmembers is a heteroatom independently selected from the group consistingof oxygen, sulfur, nitrogen, and phosphorus, and each ring in the systemcontains 3 to 7 ring members.

The term “heteroatom” means one or more of boron, oxygen, sulfur,nitrogen, phosphorus, or silicon (including, any oxidized form ofnitrogen, sulfur, phosphorus, or silicon; the quaternized form of anybasic nitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloaliphatic” and “haloalkoxy” means aliphatic or alkoxy, asthe case may be, substituted with one or more halogen atoms. The term“halogen” means F, Cl, Br, or I. Examples of haloaliphatic include—CHF₂, —CH₂F, —CF₃, —CF₂—, or perhaloalkyl, such as, —CF₂CF₃.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”. The term“aryl” also refers to heteroaryl ring systems as defined hereinbelow.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group are selected fromthe group consisting of halogen; —R^(o); —OR^(o); —SR^(o);1,2-methylene-dioxy; 1,2-ethylenedioxy; phenyl (Ph) optionallysubstituted with R^(o); —O(Ph) optionally substituted with R^(o);—(CH₂)₁₋₂(Ph), optionally substituted with R^(o); —CH═CH(Ph), optionallysubstituted with R^(o); —NO₂; —CN; —N(R^(o))₂; —NR^(o)C(O)R^(o);—NR^(o)C(O)N(R^(o))₂; —NR^(o)CO₂R^(o); —NR^(o)NR^(o)C(O)R^(o);—NR^(o)NR^(o)C(O)N(R^(o))₂; —NR^(o)NR^(o)CO₂R^(o); —C(O)C(O)R^(o);—C(O)CH₂C(O)R^(o); —CO₂R^(o); —C(O)R^(o); —C(O)N(R^(o))₂;—OC(O)N(R^(o))₂; —S(O)₂R^(o); —SO₂N(R^(o))₂; —S(O)R^(o);—NR^(o)SO₂N(R^(o))₂; —NR^(o)SO₂R^(o); —C(═S)N(R^(o))₂;—C(═NH)—N(R^(o))₂; and —(CH₂)₀₋₂NHC(O)R^(o) wherein each independentoccurrence of R^(o) is selected from the group consisting of hydrogen,optionally substituted C₁₋₆ aliphatic, an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring, phenyl, —O(Ph), and —CH₂(Ph), or,notwithstanding the definition above, two independent occurrences ofR^(o), on the same substituent or different substituents, taken togetherwith the atom(s) to which each R^(o) group is bound, form a 3-8-memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-3heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. Optional substituents on the aliphaticgroup of R^(o) are selected from the group consisting of NH₂,NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH,O(C₁₋₄aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(haloC₁₋₄aliphatic), and haloC₁₋₄aliphatic, wherein each of the foregoingC₁₋₄aliphatic groups of R^(o) is unsubstituted.

An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclicring may contain one or more substituents. Suitable substituents on thesaturated carbon of an aliphatic or heteroaliphatic group, or of anon-aromatic heterocyclic ring are selected from the group consisting ofthose listed above for the unsaturated carbon of an aryl or heteroarylgroup and additionally include the following: ═O, ═S, ═NNHR*, ═NN(R*)₂,═NNHC(O)R*, ═NNHCO₂(alkyl), ═NNHSO₂(alkyl), and ═NR*, where each R* isindependently selected from the group consisting of hydrogen and anoptionally substituted C₁₋₆ aliphatic. Optional substituents on thealiphatic group of R* are selected from the group consisting of NH₂,NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄ aliphatic, OH,O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄aliphatic), and halo(C₁₋₄ aliphatic), wherein each of the foregoingC₁₋₄aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen of a non-aromatic heterocyclicring are selected from the group consisting of —R⁺, —N(R⁺)₂, —C(O)R⁺,—CO₂R⁺, —C(O)C(O)R⁺, —C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂,—C(═NH)—N(R⁺)₂, and —NR⁺SO₂R⁺; wherein R⁺ is hydrogen, an optionallysubstituted C₁₋₆ aliphatic, optionally substituted phenyl, optionallysubstituted —O(Ph), optionally substituted —CH₂(Ph), optionallysubstituted —(CH₂)₁₋₂(Ph); optionally substituted —CH═CH(Ph); or anunsubstituted 5-6 membered heteroaryl or heterocyclic ring having one tofour heteroatoms independently selected from the group consisting ofoxygen, nitrogen, and sulfur, or, notwithstanding the definition above,two independent occurrences of R⁺, on the same substituent or differentsubstituents, taken together with the atom(s) to which each R⁺ group isbound, form a 3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroarylring having 0-3 heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. Optional substituents on thealiphatic group or the phenyl ring of R⁺ are selected from the groupconsisting of NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic),O(halo C₁₋₄ aliphatic), and halo(C₁₋₄ aliphatic), wherein each of theforegoing C₁₋₄aliphatic groups of R⁺ is unsubstituted.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of attachment to the rest of themolecule. The term “spirocycloalkylidene” refers to a carbocyclic ringthat may be fully saturated or have one or more units of unsaturationand has two points of attachment from the same ring carbon atom to therest of the molecule.

As detailed above, in some embodiments, two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein), are takentogether with the atom(s) to which each variable is bound to form a3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring having0-3 heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. Exemplary rings that are formed when twoindependent occurrences of R^(o) (or R⁺, or any other variable similarlydefined herein) are taken together with the atom(s) to which eachvariable is bound include, but are not limited to the following: a) twoindependent occurrences of R^(o) (or R⁺, or any other variable similarlydefined herein) that are bound to the same atom and are taken togetherwith that atom to form a ring, for example, N(R^(o))₂, where bothoccurrences of R^(o) are taken together with the nitrogen atom to form apiperidin-1-yl, piperazin-1-yl, or morpholin-4-yl group; and b) twoindependent occurrences of R^(o) (or R⁺, or any other variable similarlydefined herein) that are bound to different atoms and are taken togetherwith both of those atoms to form a ring, for example where a phenylgroup is substituted with two occurrences of

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) (or R⁺, or any other variablesimilarly defined herein) are taken together with the atom(s) to whicheach variable is bound and that the examples detailed above are notintended to be limiting.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays.

Description of Exemplary Compounds

In one embodiment, the present invention relates to a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

R^(N) is H, alkyl, aryl, heteroaryl, aralkyl, cycloalkyl, orheterocycloalkyl;

A is an optionally substuted 3-7 membered monocyclic ring;

B is optionally fused to a 5-7 membered ring selected from the groupconsisting of cycloaliphatic, aryl, heterocyclic, and heteroaryl;

R¹ is halo, alkyl, OH, alkoxy, trifluoromethoxy; or

two R¹ on adjacent atoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;

W is independently a bond or a (C1-C6) alkylidene chain wherein up totwo methylene units of W are independently replaced by —CO—, —O—, —S—,—SO₂—, or —NR′—;

R′ is independently H, alkyl, aryl, heteroaryl, aralkyl, cycloalkyl, orheterocyclic;

R^(W) is independently H, halo, CN, NO₂, N(R)₂, CF₃, OCF₃, OH, OR,C(O)R, CO₂R, C(O)N(R)₂, —O(C1-C6)alkylidene-OR,—O(C1-C6)alkylidene-N(R)₂, —O(C1-C6)alkylidene-heterocyclic, or anoptionally substituted aliphatic, cycloaliphatic, aryl, aryloxy,heterocyclic, or heteroaryl, wherein, when substituted, R^(w) issubstituted with up to two R²;

R² is halo, CN, NO₂, CF₃, OCF₃, OR, OC(O)R, OC(O)N(R)₂, SR, S(O)R, SO₂R,SO₂N(R)₂, SO₃R, C(O)R, CO₂R, C(O)N(R)₂, N(R)₂, NRC(O)R, NRCO₂R,NRC(O)N(R)₂, NRSO₂R, B(OR)₂, or NRSO₂N(R)₂;

R is independently H, alkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl;

n is 1 or 2;

w is an integer from 0 to 4 inclusive; and

x is an integer from 0 to 5 inclusive;

provided that when W is a bond and R^(w) is cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, —W—R^(w) is attached to the3- or 4-position of the pyridyl ring.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein A is selected from thegroup consisting of:

wherein

R³ is alkyl, alkaryl, aryl, or heteroaryl; and

q is an integer from 0 to 4 inclusive. In a further embodiment, A is

In a further embodiment, A is

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein x is 2.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein R¹ is halo. In anotherembodiment, R¹ is chloro. In another embodiment, R¹ is alkyl. In anotherembodiment, R¹ is methyl. In another embodiment, R¹ is OH. In anotherembodiment, R¹ is alkoxy. In a further embodiment, R¹ is methoxy. Inanother embodiment, R¹ is thioalkyl. In another embodiment, R¹ isthiomethyl. In a further embodiment, R¹ is trifluoromethoxy.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein two R¹ on adjacentatoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂.In a further embodiment, J is CH₂. In a further embodiment, J is CF₂.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein R^(N) is H or alkyl. Ina further embodiment, R^(N) is H. In a further embodiment, R^(N) isalkyl.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein w is 0. In a furtherembodiment, w is 1. In a further embodiment, w is 2.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein n is 1. In a furtherembodiment, n is 2.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein W is a bond. In afurther embodiment, W is an optionally substituted (C1-C6) alkylidenechain. In a further embodiment, W is —CH₂—. In a further embodiment, Wis —NH—. In a further embodiment, W is —O—. In a further embodiment, Wis —CO—. In a further embodiment, W is —OCH₂—.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein R^(w) is H. In afurther embodiment, is OH. In a further embodiment, R^(w) isheterocyclic. In a further embodiment, R^(w) is aryl. In a furtherembodiment, R^(w) is phenyl. In a further embodiment, R^(w) isheteroaryl. In a further embodiment, R^(w) is pyridyl. In a furtherembodiment, R^(w) is alkoxy. In a further embodiment, R^(w) iscycloalkyl. In a further embodiment, R^(w) is cyclohexyl.

In another embodiment, the present invention relates to a compound offormula I and the attendant definitions, wherein —W—R^(w) is acyclic. Inanother embodiment, —W—R^(w) is an aryl, heteroaryl, cycloaliphatic, orheterocycloaliphatic ring. In a further embodiment, —W(R^(w))_(n) isselected from the following:

—F, —CH₃, —CH₂CH₃, —CN, —CF₃, —CONH₂, —CH₂CH(CH₃)₂,

In a further embodiment, —W(R^(w))_(n) selected from the following:

In another embodiment, the present invention relatest to a compoundhaving formula Ia:

or a pharmaceutically acceptable salt thereof, wherein:

J is CH₂ or CF₂;

W is independently a bond or a (C1-C6) alkylidene chain wherein up totwo methylene units of W are independently replaced by —CO—, —O—, or—NR′—;

R′ is independently H or alkyl;

R^(W) is independently H, halo, CN, N(R)₂, CF₃, OH, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²;

R² is halo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂;

R is independently H, alkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl;

n is 1 or 2; and

w is an integer from 0 to 4 inclusive;

provided that when W is a bond and R^(w) is cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, is attached to the 3- or4-position of the pyridyl ring.

In another embodiment, the present invention relates to a compound offormula Ia and the attendant definitions, wherein J is CH₂. In a furtherembodiment, J is CF₂.

In another embodiment, the present invention relates to a compound offormula Ia and the attendant definitions, wherein —W—R^(w) is acyclic.In another embodiment, the present invention relates to a compound offormula Ia and the attendant definitions, wherein —W—R^(w) that is anaryl, heteroaryl, cycloaliphatic, or heterocycloaliphatic ring.

In another embodiment, the present invention relates to a compound offormula Ia and the attendant definitions, wherein —W(R^(w))_(n) isselected from the following:

—F, —CH₃, —CH₂CH₃, —CN, —CF₃, —CONH₂, —CH₂CH(CH₃)₂,

In a further embodiment, —W(R^(w))_(n) selected from the following:

In another embodiment, the present invention relates to a compoundhaving formula Ib:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is halo, alkyl, OH, alkoxy, thioalkyl, trifluoromethoxy; or

two R¹ on adjacent atoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;

W is —NR′, —CO—, or —CH₂—;

R′ is independently H or alkyl;

R^(W) is independently H, halo, CN, N(R)₂, CF₃, OH, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²;

R² is halo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂;

R is independently H, alkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl;

n is 1 or 2; and

x is an integer from 0 to 5 inclusive.

In another embodiment, the present invention relates to a compound offormula Ib and the attendant definitions, wherein two R¹ on adjacentatoms, taken together, form

and J is CH₂. In another embodiment, J is CF₂.

In another embodiment, the present invention relates to a compound offormula Ib and the attendant definitions, wherein —W(R^(w))_(n) isselected from the following:

—CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂,

In another embodiment, the present invention relates to a compoundhaving formula Ic:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is halo, alkyl, OH, alkoxy, thioalkyl, trifluoromethoxy; or

two R¹ on adjacent atoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;

W is —NR′, —CO—, or —CH₂—;

R′ is independently H or alkyl;

R^(W) is independently H, halo, CN, N(R)₂, CF₃, OH, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²;

R² is halo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂;

R is independently H, alkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl;

n is 1 or 2; and

x is an integer from 0 to 5 inclusive.

In another embodiment, the present invention relates to a compound offormula Ic and the attendant definitions, wherein two R¹ on adjacentatoms, taken together, form

and J is CH₂. In another embodiment, J is CF₂.

In another embodiment, the present invention relates to a compound offormula Ic and the attendant definitions, wherein —W(R^(w))_(n) isselected from the following:

—CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂,

In another embodiment, the present invention relates to a compound ofTable 1.

TABLE 1 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

In another embodiment, the present invention relatest to apharmaceutical composition comprising (i) a compound of the presentinvention; and (ii) a pharmaceutically acceptable carrier. In anotherembodiment, the pharmaceutical composition further comprises anadditional agent selected from the group consisting of a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, CFTR corrector, and a nutritional agent.

In another embodiment, the present invention relates to a method ofmodulating ABC transporters in a membrane of a cell, comprising the stepof contacting said cell with a compound of the present invention. Inanother embodiment, the ABC transporter is CFTR.

In another embodiment, the present invention relates to a method oftreating a condition, disease, or disorder in a patient implicated byABC transporter activity, comprising the step of administering to saidpatient a compound of the present invention. In another embodiment, theABC transporter is CFTR. In another embodiment, said condition, disease,or disorder is selected from cystic fibrosis, hereditary emphysema,hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type I hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type Ichylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, hereditary emphysema, congenital hyperthyroidism,osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency,diabetes insipidus (di), neurophyseal di, neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjögren's disease.

In another embodiment, the present invention relates to a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo, comprising: (i) a firstcomposition comprising a compound of the present invention; and (ii)instructions for: a) contacting the composition with the biologicalsample; and b) measuring activity of said ABC transporter or a fragmentthereof.

General Synthetic Schemes

Compounds of formula I can be prepared by well-known methods in the art.Illustrated below are exemplary methods for the preparation of compoundsof formula I. Schemes I below illustrates an exemplary synthetic methodfor compounds of formula I.

Synthetic Schemes

Compounds of the invention may be prepared by known methods and asillustrated in Schemes I-IV.

One of skill in the art will readily appreciate that synthetic routessuitable for various substituents of the present invention are such thatthe reaction conditions and steps.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

As discussed above, the present invention provides compounds that areuseful as modulators of ABC transporters and thus are useful in thetreatment of disease, disorders or conditions such as Cystic fibrosis,Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type I hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type I chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease (due to Prion protein processingdefect), Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjögren's disease.

Accordingly, in another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or any other adduct or derivative which uponadministration to a patient in need is capable of providing, directly orindirectly, a compound as otherwise described herein, or a metabolite orresidue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. As used herein, the term “inhibitorily activemetabolite or residue thereof” means that a metabolite or residuethereof is also an inhibitor of an ATP-Binding Cassette Transporters.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by ABC transporteractivity. In certain embodiments, the present invention provides amethod of treating a condition, disease, or disorder implicated by adeficiency of ABC transporter activity, the method comprisingadministering a composition comprising a compound of formula (I) to asubject, preferably a mammal, in need thereof.

In certain preferred embodiments, the present invention provides amethod of treating cystic fibrosis, hereditary emphysema (due toa1-antitrypsin; non Piz variants), hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type I hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to ΔAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome, comprising the step of administering to saidmammal an effective amount of a composition comprising a compound offormula (I), or a preferred embodiment thereof as set forth above.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of formula (I), or a preferred embodiment thereofas set forth above.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of cystic fibrosis,hereditary emphysema (due to α1-antitrypsin; non Piz variants),hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of cystic fibrosis, hereditary emphysema (due to α1-antitrypsin;non Piz variants), hereditary hemochromatosis, coagulation-fibrinolysisdeficiencies, such as protein C deficiency, Type 1 hereditaryangioedema, lipid processing deficiencies, such as familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,mucopolysaccharidoses (due to lysosomal processing enzymes),Sandhof/Tay-Sachs (due to β-hexosaminidase), Crigler-Najjar type II (dueto UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used to,increase the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As described generally above, the compounds of the invention are usefulas modulators of ABC transporters. Thus, without wishing to be bound byany particular theory, the compounds and compositions are particularlyuseful for treating or lessening the severity of a disease, condition,or disorder where hyperactivity or inactivity of ABC transporters isimplicated in the disease, condition, or disorder. When hyperactivity orinactivity of an ABC transporter is implicated in a particular disease,condition, or disorder, the disease, condition, or disorder may also bereferred to as a “ABC transporter-mediated disease, condition ordisorder”. Accordingly, in another aspect, the present inventionprovides a method for treating or lessening the severity of a disease,condition, or disorder where hyperactivity or inactivity of an ABCtransporter is implicated in the disease state.

The activity of a compound utilized in this invention as a modulator ofan ABC transporter may be assayed according to methods describedgenerally in the art and in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, aminone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound disclosed inWO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740.

In another embodiment, the additiona agent is a benzo(c)quinoliziniumderivative that exhibits CFTR modulation activity or a benzopyranderivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed inU.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.

In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006002421,WO2006099256, WO2006127588, or WO2007044560.

In another embodiment, the an additional agent selected from compoundsdisclosed in U.S. patent application Ser. No. 11/165,818, published asU.S. Published Patent Application No. 2006/0074075, filed Jun. 24, 2005,and hereby incorporated by reference in its entirety. In anotherembodiment, the additional agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating ABC transporteractivity in a biological sample or a patient (e.g., in vitro or invivo), which method comprises administering to the patient, orcontacting said biological sample with a compound of formula I or acomposition comprising said compound. The term “biological sample”, asused herein, includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Modulation of ABC transporter activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, the study ofABC transporters in biological and pathological phenomena; and thecomparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formula (I). In preferredembodiments, the anion channel is a chloride channel or a bicarbonatechannel. In other preferred embodiments, the anion channel is a chloridechannel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of formula (I). The term “functional ABC transporter” as usedherein means an ABC transporter that is capable of transport activity.In preferred embodiments, said functional ABC transporter is CFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential. Means for measuring the voltage potential across a membranein the biological sample may employ any of the known methods in the art,such as optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo comprising (i) a compositioncomprising a compound of formula (I) or any of the above embodiments;and (ii) instructions for a) contacting the composition with thebiological sample and b) measuring activity of said ABC transporter or afragment thereof. In one embodiment, the kit further comprisesinstructions for a) contacting an additional composition with thebiological sample; b) measuring the activity of said ABC transporter ora fragment thereof in the presence of said additional compound, and c)comparing the activity of the ABC transporter in the presence of theadditional compound with the density of the ABC transporter in thepresence of a composition of formula (I). In preferred embodiments, thekit is used to measure the density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Preparation of 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylicacid

A mixture of 2-(benzo[d][1,3]dioxol-5-yl)acetonitrile (5.10 g 31.7mmol), 1-bromo-2-chloro-ethane (9.00 mL 109 mmol), andbenzyltriethylammonium chloride (0.181 g, 0.795 mmol) was heated at 70°C. and then 50% (wt./wt.) aqueous sodium hydroxide (26 mL) was slowlyadded to the mixture. The reaction was stirred at 70° C. for 24 hoursand then heated at 130° C. for 48 hours. The dark brown reaction mixturewas diluted with water (400 mL) and extracted once with an equal volumeof ethyl acetate and once with an equal volume of dichloromethane. Thebasic aqueous solution was acidified with concentrated hydrochloric acidto pH less than one and the precipitate filtered and washed with 1 Mhydrochloric acid. The solid material was dissolved in dichloromethane(400 mL) and extracted twice with equal volumes of 1 M hydrochloric acidand once with a saturated aqueous solution of sodium chloride. Theorganic solution was dried over sodium sulfate and evaporated to drynessto give a white to slightly off-white solid (5.23 g, 80%) ESI-MS m/zcalc. 206.1, found 207.1 (M+1)⁺. Retention time 2.37 minutes. ¹H NMR(400 MHz, DMSO-d₆) δ 1.07-1.11 (m, 2H), 1.38-1.42 (m, 2H), 5.98 (s, 2H),6.79 (m, 2H), 6.88 (m, 1H), 12.26 (s, 1H).

Preparation of1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid

Step a: 2,2-Difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester

A solution of 5-bromo-2,2-difluoro-benzo[1,3]dioxole (11.8 g, 50.0 mmol)and tetrakis(triphenylphosphine)palladium (0) [Pd(PPh₃)₄, 5.78 g, 5.00mmol] in methanol (20 mL) containing acetonitrile (30 mL) andtriethylamine (10 mL) was stirred under a carbon monoxide atmosphere (55PSI) at 75° C. (oil bath temperature) for 15 hours. The cooled reactionmixture was filtered and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography to give crude2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester (11.5 g),which was used directly in the next step.

Step b: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-methanol

Crude 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester(11.5 g) dissolved in 20 mL of anhydrous tetrahydrofuran (THF) wasslowly added to a suspension of lithium aluminum hydride (4.10 g, 106mmol) in anhydrous THF (100 mL) at 0° C. The mixture was then warmed toroom temperature. After being stirred at room temperature for 1 hour,the reaction mixture was cooled to 0° C. and treated with water (4.1 g),followed by sodium hydroxide (10% aqueous solution, 4.1 mL). Theresulting slurry was filtered and washed with THF. The combined filtratewas evaporated to dryness and the residue was purified by silica gelcolumn chromatography to give(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol, 76% overtwo steps) as a colorless oil.

Step c: 5-Chloromethyl-2,2-difluoro-benzo[1,3]dioxole

Thionyl chloride (45 g, 38 mmol) was slowly added to a solution of(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol) indichloromethane (200 mL) at 0° C. The resulting mixture was stirredovernight at room temperature and then evaporated to dryness. Theresidue was partitioned between an aqueous solution of saturated sodiumbicarbonate (100 mL) and dichloromethane (100 mL). The separated aqueouslayer was extracted with dichloromethane (150 mL) and the organic layerwas dried over sodium sulfate, filtrated, and evaporated to dryness togive crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g) whichwas used directly in the next step.

Step d: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile

A mixture of crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g)and sodium cyanide (1.36 g, 27.8 mmol) in dimethylsulfoxide (50 mL) wasstirred at room temperature overnight. The reaction mixture was pouredinto ice and extracted with ethyl acetate (300 mL). The organic layerwas dried over sodium sulfate and evaporated to dryness to give crude(2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile (3.3 g) which was useddirectly in the next step.

Step e: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile

Sodium hydroxide (50% aqueous solution, 10 mL) was slowly added to amixture of crude (2,2-difluoro-benzo[1,3]dioxol-5-yl-acetonitrile,benzyltriethylammonium, chloride (3.00 g, 15.3 mmol), and1-bromo-2-chloroethane (4.9 g, 38 mmol) at 70° C. The mixture wasstirred overnight at 70° C. before the reaction mixture was diluted withwater (30 mL) and extracted with ethyl acetate. The combined organiclayers were dried over sodium sulfate and evaporated to dryness to givecrude 1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile,which was used directly in the next step.

Step f: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylicacid

1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile (crudefrom the last step) was refluxed in 10% aqueous sodium hydroxide (50 mL)for 2.5 hours. The cooled reaction mixture was washed with ether (100mL) and the aqueous phase was acidified to pH 2 with 2M hydrochloricacid. The precipitated solid was filtered to give1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid as awhite solid (0.15 g, 1.6% over four steps). ESI-MS m/z calc. 242.04,found 241.58 (M+1)⁺; ¹H NMR (CDCl₃) δ 7.14-7.04 (m, 2H), 6.98-6.96 (m,1H), 1.74-1.64 (m, 2H), 1.26-1.08 (m, 2H).

The following Table 2 contains a list of carboxylic acid building blocksthat were commercially available, or prepared by the methods describedabove.

TABLE 2 Carboxylic acid building blocks Name Structure1-(2-methoxyphenyl) cyclopropane-1- carboxylic acid

1-(3-methoxyphenyl) cyclopropane-1- carboxylic acid

1-(4-methoxyphenyl) cyclopropane-1- carboxylic acid

1-[4- (trifluoromethoxy) phenyl]cyclopropane-1- carboxylic acid

1-phenylcyclopropane- 1-carboxylic acid

1-(4-chlorophenyl) cyclopropane-1- carboxylic acid

1-(3,4- dimethoxyphenyl) cyclopropanecarboxylic acid

1-(4- (methylthio)phenyl) cyclopropanecarboxylic acid

1-p-tolylcyclo- propanecarboxylic acid

Preparation of (6-aminopyridin-3-yl)(2-methoxyphenyl)methanone

Step a: 6-chloro-N-methoxy-N-methyl-nicotinamide

To a solution of 6-chloro-nicotinic acid (94.5 g, 0.6 mol) indichloromethane (1000 mL) was added N-methyl morpholine (181.8 g, 1.8mol) followed by iso-butyl chloroformate (81.9 g, 0.6 mol) at −25° C.under 1\2 atmosphere. The mixture was stirred for 15 minutes and thenO,N-dimethyl hydroxylamine hydrochloride (64.35 g, 0.66 mol) was added.The mixture was stirred for 30 min at −25° C. and warmed to roomtemperature slowly. The mixture was stirred for an additional hour atroom temperature and a saturated solution of NaHCO₃ (800 mL) was added.The separated aqueous layer was extracted with dichloromethane (500mL×3). The combined organic layers were washed with water and brine. Thesolvent was evaporated to give the crude product, which was purified bycolumn chromatography to give the pure product6-chloro-N-methoxy-N-methyl-nicotinamide as a white solid (56 g, 46.6%).

Step b: (6-chloropyridin-3-yl)-(2-methoxyphenyl)-methanone

To a solution of 1-bromo-2-methoxy-benzene (49.2 g, 0.263 mol) in dryTHF (500 mL) was added dropwise n-BuLi (2.5 M in hexane, 105 mL, 0.263mol) at −78° C. under N₂ atmosphere. The mixture was stirred at thistemperature for 1 hr and then a solution of6-chloro-N-methoxy-N-methyl-nicotinamide (50 g, 0.25 mol) in THF (100mL) was added dropwise. The reaction mixture was warmed to roomtemperature, stirred overnight and then was quenched with a saturatedsolution of NH₄Cl (300 mL). The separated aqueous layer was extractedwith ethyl acetate (3×300 mL). The combined organic layers were washedwith brine, dried over Na₂SO₄, evaporated in vacuo to give the crudeproduct that was purified by column chromatography to yield(6-chloropyridin-3-yl)-(2-methoxyphenyl)-methanone as a white solid (31g, 47.6%).

Step c: (6-Amino-pyridin-3-yl)-(2-methoxyphenyl)-methanone

A solution of 6-chloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone (30 g,0.121 mol) in ethanol (150 ml) and anhydrous ammonia (100 ml) was placedin a bomb and heated at 145° C. for 16 hours. The solvents were removedin vacuo and the residue was purified by column chromatography to give(6-Amino-pyridin-3-yl)-(2-methoxyphenyl)-methanone (18 g, 65.2%). ¹H NMR(DMSO-d6): δ: 8.12 (s, 1H), 7.68 (d, J=8.8 Hz, 1H), 7.47 (t, J=7.2, 1H),7.21 (d, J=7.2, 1H), 7.13 (d, J=7.2, 1H), 7.03 (d, 1H), 6.96 (br, 2H),6.44 (d, J=8.8, 1H), 3.69 (s, 3H). MS (ESI) m/e (M+H⁺) 229.27.

Preparation of 5-(2-methoxybenzyl)pyridin-2-amine

Step a: (6-aminopyridin-3-yl)-(2-methoxyphenyl)-methanol

To a solution of (6-amino-pyridin-3-yl)-(2-methoxy-phenyl)-methanone (14g, 61.4 mol) in CH₃OH (150 mL) was added NaBH₄ (3.48 g, 92.1 mmol)portion-wise at 0° C. After the addition was completed, the reaction waswarmed to room temperature and stirred at room temperature for 1 hr. Thesolvent was removed in vacuo and water (100 mL) was added to theresidue. The mixture was extracted with dichloromethane (100 mL×3). Thecombined organic layers were washed with saturated solution of NaHCO₃and brine, dried over Na₂SO₄, filtered and concentrated in vacuo to givea crude product that was used directly in the next step with no furtherpurification.

Step b: 5-(2-methoxy-benzyl)-pyridin-2-ylamine

A mixture of the crude compound from previous step, Et₃SiH (57 g, 491mmol) and TFA (112 g, 982 mmol) in CH₂Cl₂ (150 mL) was stirred at roomtemperature overnight. The excess Et₃SiH, TFA and CH₂Cl₂ were removedunder reduced pressure. The residue was stirred in water; the solidprecipitate was filtered, washed with diethyl ether and petroleum etherto give 5-(2-methoxy-benzyl)-pyridin-2-ylamine as TFA salt. (5 g, 24.8%for 2 steps). ¹H NMR (DMSO-d₆): δ 13.5 (br, 1H), 7.93 (brs, 2H),7.75-7.73 (m, 2H), 7.22 (t, J=8.0 Hz, 1H), 7.19 (d, J=7.2 Hz, 1H), 6.98(d, J=8.0 Hz, 1H), 6.90-6.86 (m, 2H), 3.77 (s, 2 H), 3.76 (s, 3H). MS(ESI) m/e (M+H⁺) 215.27.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)cyclopropanecarboxamide

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (41 mg, 0.20 mmol)was placed in an oven-dried flask under nitrogen. Thionyl chloride (0.3mL) and N,N-dimethylformamide (0.03 mL) were added and the solution wasallowed to stir for 10 minutes at room temperature. The excess thionylchloride was removed under vacuum and the resulting solid was suspendedin anhydrous pyridine (1 mL). This solution was then slowly added to asolution of pyridin-2-amine (19 mg, 0.20 mmol) in anhydrous pyridine (1mL). The resulting mixture was allowed to stir for 15 hours at 110° C.The crude product was then filtered, evaporated to dryness, dissolved inN,N-dimethylformamide (1 mL) and purified by reverse-phase preparativeliquid chromatography utilizing a gradient of 0-99% acetonitrile inwater containing 0.05% trifluoroacetic acid to yield the pure product(5.9 mg, 0.021 mmol, 10%). ESI-MS m/z calc. 282.1, found 283.1 (M+1)⁺.Retention time 2.13 minutes.

Preparation of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(5-methylpyridin-2-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid(2.32 g, 9.58 mmol) was placed in an oven-dried flask under nitrogen.Thionyl chloride (3 mL) and N,N-dimethylformamide (0.3 mL) were addedand the solution was allowed to stir for 30 minutes at room temperature.The excess thionyl chloride was removed under vacuum and the resultingsolid was suspended in anhydrous dichloromethane (10 mL). This solutionwas then slowly added to a solution 5-methylpyridin-2-amine (0.798 g,7.38 mmol) in anhydrous dichloromethane (10 mL) containing triethylamine(4.11 mL, 29.5 mmol). The resulting mixture was allowed to stir for 15hours at room temperature. The crude product was then washed two timeswith a 1M aqueous solution of hydrochloric acid, two times with asaturated aqueous solution of sodium bicarbonate, and finally two timeswith a saturated aqueous solution of sodium chloride. The organic layerwas dried over sodium sulfate, evaporated to near dryness, and thenpurified on 120 g of silica gel utilizing a gradient of 0-30% ethylacetate in hexanes to yield the pure product (1.20 g, 3.60 mmol, 48.7%).ESI-MS m/z calc. 332.1, found 333.1 (M+1)⁺. Retention time 1.48 minutes.

The compounds listed in Table 3 below were made in a manner analogous tothat described above from the appropriate reactants:

TABLE 3 Name Structure 1-(benzo[d][1,3]dioxol-5-yl)-N-(6-methylpyridin-2- yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- methylpyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- cyanopyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(4- ethylpyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(4- methylpyridin-2-yl)cyclopropanecarboxamide

6-(1-(benzo[d][1,3]dioxol-5- yl)cyclopropanecarboxamido)nicotinamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- (trifluoromethyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(3-ethyl-6- methylpyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(6- ethylpyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- fluoropyridin-2-yl)cyclopropanecarboxamide

N-(5-(2-methoxybenzyl)pyridin-2-yl)-1- (2-methoxyphenyl)cyclopropanecarboxamide

N-(5-(3-methoxybenzyl)pyridin-2-yl)-1- (2-methoxyphenyl)cyclopropanecarboxamide

N-(3-(benzyloxy)pyridin-2-yl)-1- phenylcyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(5-methylpyridin- 2-yl)cyclopropanecarboxamide

N-(6-ethylpyridin-2-yl)-1- phenylcyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(3- (benzyloxy)pyridin-2-yl)cyclopropanecarboxamide

N-(4-ethylpyridin-2-yl)-1-(4- methoxyphenyl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(6-methylpyridin- 2-yl)cyclopropanecarboxamide

N-(6-ethylpyridin-2-yl)-1-(4- methoxyphenyl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(3-ethyl-6- methylpyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- fluoropyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5- methylpyridin-2-yl)cyclopropanecarboxamide

Preparation ofN-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamide

1-(4-Methoxyphenyl)cyclopropanecarboxylic acid (19.2 mg, 0.100 mmol) and(6-aminopyridin-3-yl)(2-methoxyphenyl)methanone (22.8 g, 0.100 mmol)were dissolved in N,N-dimethylformamide (DMF, 0.7 mL) containingtriethylamine (0.042 mL, 0.300 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 38.0 mg, 0.100 mmol) was added and thesolution was allowed to stir for 72 hours at 70° C. The mixture was thenpurified by reverse phase preparative liquid chromatography utilizing agradient of 0-99% acetonitrile in water containing 0.05% trifluoroaceticacid to yield the pure product (4.2 mg, 0.010 mmol, 10%). ESI-MS m/zcalc. 402.2, found 403.5 (M+1)⁺. Retention time 3.50 minutes.

The compounds listed in Table 4 below were made in a manner analogous tothat ofN-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamidefrom the appropriate reactants:

TABLE 4 Name Structure N-(5-(2-methoxybenzyl)pyridin-2-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(2- methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(2- methoxybenzoyl)pyridin-2-yl)cyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamide

N-(5-(2-methoxybenzyl)pyridin-2-yl)-1-p- tolylcyclopropanecarboxamide

1-(4-chlorophenyl)-N-(5-(2-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(3,4-dimethoxyphenyl)-N-(5-(2- methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(3-methoxyphenyl)-N-(5-(2- methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

N-(5-(2-methoxybenzyl)pyridin-2-yl)-1-(4-(trifluoromethoxy)phenyl)cyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1- phenylcyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-p- tolylcyclopropanecarboxamide

1-(4-chlorophenyl)-N-(5-(2- methoxybenzoyl)pyridin-2-yl)cyclopropanecarboxamide

1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(5-(2-methoxybenzoyl)pyridin-2- yl)cyclopropanecarboxamide

1-(3,4-dimethoxyphenyl)-N-(5-(2- methoxybenzoyl)pyridin-2-yl)cyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(2-methoxyphenyl)cyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(4-(methylthio)phenyl)cyclopropanecarboxamide

N-(5-(2-methoxybenzoyl)pyridin-2-yl)-1-(4-(trifluoromethoxy)phenyl)cyclopropanecarboxamide

Preparation of3-(2-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-5-methylpyridin-4-yl)benzoicacid)

Step a: tert-butyl 3-(3-methylpyridin-4-yl)benzoate

4-Chloro-3-methylpyridine (2.00 g, 15.7 mmol),3-(tert-butoxycarbonyl)phenylboronic acid (5.42 g, 24.4 mmol), a 2Maqueous solution of potassium carbonate (31.4 mL, 62.8 mmol), andpalladium tetrakis(triphenylphosphine) (Pd(PPh₃)₄, 0.906 g, 0.784 mmol)were suspended in 1,2-dimethoxyethane (DME, 150 mL). The resultingmixture was stirred and heated to 80° C. for 60 hours. The crudereaction mixture was cooled to room temperature and then the layers wereseparated. The organic layer was evaporated to dryness and then purifiedon 120 g of silica gel utilizing a gradient of 0-70% ethyl acetate inhexanes to yield the pure product as a pale yellow oil (3.02 g, 11.2mmol, 71.4%).

Step b: 4-(3-(tert-Butoxycarbonyl)phenyl)-3-methylpyridine 1-oxide

tert-Butyl 3-(3-methylpyridin-4-yl)benzoate (0.500 g, 1.86 mmol) wasdissolved in a mixture of dichloromethane (0.800 mL) and 30% hydrogenperoxide (0.421 mL). Methyltrioxorhenium (VII) (2.3 mg, 0.92 mmol) wasadded and the reaction mixture was stirred vigorously for 5 hours. Thelayers were then separated and the organic layer was treated with sodiumsulfite, and then dried over sodium sulfate. The crude product filtered,evaporated to dryness, and used without further purification. ESI-MS m/zcalc. 285.1, found 286.1 (M+1)⁺. Retention time 1.22 minutes.

Step c: tert-Butyl 3-(2-amino-5-methylpyridin-4-yl)benzoate

4-(3-(tert-Butoxycarbonyl)phenyl)-3-methylpyridine 1-oxide (0.467 g,1.64 mmol) was dissolved in a mixture of pyridine (0.53 mL) andacetonitrile (15 mL) under an atmosphere of argon.4-Methylbenzene-1-sulfonyl chloride (0.406 g, 2.13 mmol) was added andthe reaction mixture was stirred at 75° C. for 72 hours. Ethanolamine (7mL) was then added and the reaction mixture was allowed to stir for 5minutes at room temperature. The crude product was partitioned betweendichloromethane and a saturated aqueous solution of sodium bicarbonate.The layers were separated and the organic layer was washed with asaturated aqueous solution of sodium chloride. The organic layer wasdried over sodium sulfate and then purified on 40 g of silca gelutilizing a gradient of 0-100% ethyl acetate in hexanes to yield thepure product (0.169 g, 0.594 mmol, 36.3%). ESI-MS m/z calc. 284.2, found285.1 (M+1)⁺. Retention time of 1.28 minutes. ¹H NMR (400.0 MHz,DMSO-d₆) δ 7.94-7.91 (m, 1H), 7.84 (s, 1H), 7.79 (d, J=1.7 Hz, 1H),7.62-7.56 (m, 2H), 6.33 (s, 1H), 5.78 (s, 2H), 1.99 (s, 3H), 1.55 (s,9H).

Step d: tert-Butyl3-(2-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-5-methylpyridin-4-yl)benzoate

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid (144mg, 0.593 mmol) was placed in an oven-dried flask under nitrogen.Thionyl chloride (1 mL) and N,N-dimethylformamide (0.1 mL) were addedand the solution was allowed to stir for 30 minutes at room temperature.The excess thionyl chloride was removed under vacuum and the resultingsolid was suspended in 2 mL of anhydrous dichloromethane. This solutionwas then slowly added to a solution of tert-butyl3-(2-amino-5-methylpyridin-4-yl)benzoate (129 mg, 0.454 mmol) in 5 mL ofanhydrous dichloromethane containing triethylamine (0.165 mL, 1.19mmol). The resulting mixture was allowed to stir for 15 hours at roomtemperature. The crude product was evaporated to dryness and thenpurified on 12 g of silica gel utilizing a gradient of 0-40% ethylacetate in hexanes to yield the pure product as a yellow solid (162 mg,0.319 mmol, 70.3%). ESI-MS m/z calc. 508.2, found; 509.1 (M+1)⁺Retention time 2.22 minutes. ¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (s, 1H),8.18 (s, 1H), 7.98-7.96 (m, 1H), 7.89 (s, 1H), 7.82 (d, J=1.6 Hz, 1H),7.64-7.62 (m, 2H), 7.57 (d, J=1.6 Hz, 1H), 7.42 (d, J=8.3 Hz, 1H), 7.34(dd, J=1.7, 8.3 Hz, 1H), 2.14 (s, 3H), 1.55 (s, 9H), 1.51-1.49 (m, 2H)1.19-1.15 (m, 2H).

Step e:3-(2-O-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-5-methylpyridin-4-yl)benzoicacid

tert-Butyl3-(2-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-5-methylpyridin-4-yl)benzoate(40. mg, 0.079 mmol) was dissolved in dichloromethane (1 mL).Trifluoroacetic acid (0.500 mL) was added and the reaction mixture wasstirred at room temperature for 1 hour. The crude reaction mixture wasevaporated to dryness to yield the pure product. ESI-MS m/z calc. 452.1,found; 453.0 (M+1)⁺ Retention time 1.64 minutes. ¹H NMR (400 MHz,DMSO-d₆) δ 1H NMR (400.0 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.22 (s, 1H),8.03-8.00 (m, 1H), 7.89 (d, J=2.5 Hz, 2H), 7.68-7.64 (m, 2H), 7.58 (d,J=1.6 Hz, 1H), 7.43 (d, J=8.3 Hz, 1H), 7.35 (dd, J=1.7, 8.3 Hz, 1H),2.18 (s, 3H), 1.54-1.51 (m, 2H), 1.21-1.17 (m, 2H).

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(cyclohexylmethyl)pyridin-2-yl)cyclopropanecarboxamide

Step a:1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide

To 1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid (2.4 g, 11.6mmol) in thionyl chloride (2.5 mL, 34.7 mmol) was addedN,N-dimethylformamide (0.1 mL). The reaction mixture was stirred at roomtemperature for 30 minutes before it was evaporated to dryness to yieldthe acid chloride.

To 6-bromopyridin-2-amine (2.0 g, 11.6 mmol) in pyridine (10 mL) wasadded the acid chloride. The reaction was heated to 100° C. for 12hours. The reaction was diluted with dichloromethane (30 mL) and washedwith 1 N NaOH (3×20 mL). The organics were dried over Na₂SO₄ andevaporated to dryness. The crude material was purified by silica gelchromatography (eluting with 0-50% ethyl acetate in hexanes) to yieldthe product (2.9 g, 8.0 mmol, 70%). ESI-MS m/z calc. 360.01, found 361.1(M+1)⁺. Retention time 3.59 minutes.

Step b:1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(cyclohexylmethyl)pyridin-2-yl)cyclopropanecarboxamide

To a 0.5 M solution of (cyclohexylmethyl)zinc(II) bromide in THF (0.8mL, 0.4 mmol) was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (8 mg, 0.01mmol) and the reaction was stirred under nitrogen for 20 minutes.1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide(36 mg, 0.1 mmol) was added and the reaction was irradiated in themicrowave for 10 minutes at 150° C. The reaction was quenched with asaturated ammonium chloride solution (2 mL) and a saturatedethylenediamine tetraacetic acid disodium salt solution (2 mL). Themixture was stirred for 30 minutes before it was extracted withdichloromethane (3×4 mL). The organics were dried over Na₂SO₄ andevaporated. The crude product was dissolved in DMSO (1 mL) and purifiedby reverse-phase preparative liquid chromatography utilizing a gradientof 0-99% acetonitrile in water containing 0.05% trifluoracetic acid toyield the pure product (20 mg, 0.05 mmol, 50%). ESI-MS m/z calc. 378.19,found 379.1 (M+1)⁺; retention time 3.55 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-isobutylpyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-isobutylpyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamideand isobutylzinc(II) chloride in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(cyclohexylmethyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation ofN-(6-(benzylamino)-5-methylpyridin-2-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

Step a:N-(6-chloro-5-methylpyridin-2-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

To 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid(18.8 g, 78 mmol) in thionyl chloride (17 mL, 233 mmol) was addedN,N-dimethylformamide (0.2 mL, 2.6 mmol). The reaction mixture wasstirred at room temperature for two hours. Excess thionyl chloride andN,N-dimethylformamide were removed in vacuo and the resulting acidchloride was used directly in next step.

To a solution of 6-chloro-5-methylpyridin-2-amine (11.1 g, 78 mmol) andtriethylamine (22.0 mL, 156 mmol) in dichloromethane (100 mL) was addeda solution of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride(20.3 g, 78 mmol) in dichloromethane (50 mL). The resulting reactionmixture was allowed to stir at room temperature for eighteen hours. Thereaction mixture was then washed with 1N aqueous NaOH (2×200 mL), 1 Naqueous HCl (1×200 mL) and saturated aqueous NaHCO₃ (1×200 mL). Theorganics were dried over sodium sulfate and evaporated to yield theproduct (26.9 g, 73.3 mmol, 94% 2-step). ESI-MS m/z calc. 366.06, found367.3 (M+1)⁺. Retention time 2.19 minutes. ¹H NMR (400 MHz, DMSO-d₆) δ9.30 (s, 1H), 7.89-7.87 (m, 1H), 7.78-7.76 (m, 1H), 7.54-7.53 (m, 1H),7.41-7.39 (m, 1H), 7.33-7.30 (m, 1H), 2.26 (s, 3H), 1.52-1.49 (m, 2H),1.19-1.16 (m, 2H).

Step b.N-(6-(benzylamino)-5-methylpyridin-2-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

To benzylamine (0.5 mL, 4.6 mmol) was addedN-(6-chloro-5-methylpyridin-2-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(37 mg, 0.1 mmol) and the reaction mixture was irradiated in themicrowave at 170° C. for 60 minutes. The crude product purified byreverse-phase preparative liquid chromatography utilizing a gradient of0-99% acetonitrile in water containing 0.05% trifluoracetic acid toyield the pure product. ESI-MS m/z calc. 437.4, found 438.3 (M+1)⁺;retention time 1.83 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(2-methoxyphenylamino)pyridin-2-yl)cyclopropanecarboxamide

To1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide(72 mg, 0.2 mmol), XANTPHOS (7.00 mg, 0.008 mmol), K^(t)BuO (31 mg, 0.28mmol), (DPPF)₂PdCl₂.CH₂Cl₂ (33.00 mg, 0.24 mmol), and 2-methoxyaniline(30 mg, 0.24 mmol), 1,4-dioxane (0.400 mL) and triethylamine (0.200 mL)were added. The reaction mixture was heated to 150° C. in a microwavereactor for 10 minutes. The resulting material was cooled to roomtemperature. The solvent was evaporated under reduced pressure. Theresulting mixture was dissolved in dichloromethane and washed with H₂O.The organic layer was dried over anhydrous Na₂SO₄ and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel to yield1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(2-methoxyphenylamino)pyridin-2-yl)cyclopropanecarboxamidewhich was then treated with HCl in MeOH to form the HCl salt (2.4 mg,0.0055 mmol, 2.7%). ESI-MS m/z calc. 403.1, found 404.5 (M+1)⁺;retention time 3.01 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-((2-methoxyphenyl)(methyl)amino)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-((2-methoxyphenyl)(methyl)amino)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(2-methoxyphenylamino)pyridin-2-yl)cyclopropanecarboxamideby reacting 2-methoxy-N-methylaniline with1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzoylpyridin-2-yl)cyclopropanecarboxamide

To a solution of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-cyanopyridin-2-yl)cyclopropanecarboxamide(61.00 mg, 0.20 mmol) in THF (1 mL) at 0° C., a solution ofphenylmagnesium bromide (133 μL, 3 M, 0.40 mmol) in ether was slowlyadded and the reaction mixture was allowed to warm up to roomtemperature and stirred at room temperature for eighty hours.Hydrochloric acid (1M) was added to the reaction material. The productwas extracted using ethyl acetate. The organic layer was dried overanhydrous Na₂SO₄ and evaporated under reduced pressure. The crudeproduct was dissolved in DMSO (1 mL), filtered and purified by reversephase preparative HPLC to yield1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzoylpyridin-2-yl)cyclopropanecarboxamideas a TFA salt (18 mg, 0.035 mmol, 18%). ESI-MS m/z calc. 386.1, found387.1 (M+1)⁺; retention time 3.45 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

To (DPPF)₂PdCl₂.CH₂Cl₂ (17 mg, 0.02 mmol), a solution of(4-methoxybenzyl)zinc(II) chloride (1.60 mL, 0.5 M, 0.80 mmol) in THFwas added under nitrogen atmosphere and stirred at room temperature forfive minutes. To this, a solution of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide(72 mg, 0.20 mmol) in THF (800 μL) was added slowly under nitrogenatmosphere. The reaction was heated to 150° C. in a microwave reactorfor 10 minutes. The resulting material was cooled to room temperature.Na₂EDTA and saturated aqueous NH₄Cl were added to the reaction materialand stirred at room temperature for 30 minutes. The product wasextracted using dichloromethane. The organic layer was dried overanhydrous Na₂SO₄ and evaporated under reduced pressure. The crudeproduct was dissolved in DMSO (1 mL), filtered and purified by reversephase preparative HPLC to yield1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamideas a TFA salt (27 mg, 0.052 mmol, 26%). ESI-MS m/z calc. 402.2, found403.0 (M+1)⁺; retention time 3.24 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(2-chlorobenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(2-chlorobenzyl)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamideby reacting (2-chlorobenzyl)zinc(II) chloride with1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(2-methylbenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(2-methylbenzyl)pyridin-2-yl)cyclopropanecarboxamide(VRT-810361) was synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamideby reacting (2-methylbenzyl)zinc(II) chloride with1-(benzo[d][1,3]dioxol-5-yl)-N-(6-bromopyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]-dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamide

Step a:1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamide

To 1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid (9.50 g,46.00 mmol), thionyl chloride (10.00 mL, 138.00 mmol) and DMF (4 drops)were added and the reaction mixture was stirred and heated at 60° C. forthirty minutes. The excess thionyl chloride was evaporated under reducedpressure. A portion of the acid chloride (14.4 mmol) was dissolved inpyridine (10 mL) and was slowly added to 5-bromopyridin-2-amine (14.4mmol) in pyridine (10 mL). The reaction mixture was stirred at 110° C.for one hour and thirty minutes. The pyridine was evaporated underreduced pressure. The resulting mixture was dissolved in dichloromethane(100 mL) and washed with 1 N NaOH (3×25 mL). The organic layer was driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel to yield1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamide(4.20 g, 11.7 mmol, 81%).

Step b:1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamide

A solution of benzylzinc(II) bromide (1.60 mL, 0.5 M, 0.8 mmol) in THFand (DPPF)₂PdCl₂.CH₂Cl₂ (17 mg, 0.02 mmol) was stirred at roomtemperature for 20 minutes under nitrogen atmosphere. To this, asolution of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamide(72 mg, 0.20 mmol) in THF (800 μL) was added and the reaction mixturewas heated to 150° C. in a microwave reactor for 10 minutes. Theresulting material was cooled to room temperature. Na₂EDTA and saturatedaqueous NH₄Cl were added to the reaction material and stirred at roomtemperature for 20 minutes. The product was extracted usingdichloromethane. The organic layer was dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The crude product was dissolved inDMSO (1 mL), filtered and purified by reverse phase preparative HPLC toyield1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideas a TFA salt (15 mg, 0.031 mmol, 15%). ESI-MS m/z calc. 372.1, found373.1 (M+1)⁺; retention time 7.14 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(2,6-dichlorobenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2,6-dichlorobenzyl)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideby reacting1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamidewith (2,6-dichlorobenzyl)zinc(II) chloride.

Preparation of1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(cyclohexylmethyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(cyclohexylmethyl)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideby reacting1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamidewith (cyclohexylmethyl)zinc(II) bromide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-isobutylpyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-isobutylpyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideby reacting1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamidewith isobutylzinc(II) bromide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-methoxybenzyl)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideby reacting1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamidewith (4-methoxybenzyl)zinc(II) chloride.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(2-chlorobenzyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2-chlorobenzyl)pyridin-2-yl)cyclopropanecarboxamidewas synthesized using the procedure of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-benzylpyridin-2-yl)cyclopropanecarboxamideby reacting1-(benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropanecarboxamidewith (2-chlorobenzyl)zinc(II) chloride.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenylamino)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-iodopyridin-2-yl)cyclopropanecarboxamide(816 mg, 0.200 mmol) was suspended in 1,4-dioxane (0.4 mL) andtriethylamine (0.2 mL) containing cesium carbonate (91.2 mg, 0.0280mmol), tris(dibenzylideneacetone)dipalladium (0) (1.8 mg, 0.0020 mmol),2-methoxyaniline (0.027 mL, 0.24 mmol), and(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (XANTPHOS, 2.3mg, 0.0040 mmol). The reaction mixture was heated to 100° C. for 15hours. The crude product was then filtered, evaporated to dryness,dissolved in N,N-dimethylformamide (1 mL) and purified by reverse-phasepreparative liquid chromatography utilizing a gradient of 0-99%acetonitrile in water containing 0.05% trifluoroacetic acid to yield thepure product (2.6 mg, 0.0049, 2.4%). ESI-MS m/z calc. 403.2, found 404.5(M+1)⁺. Retention time 2.73 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2-methoxybenzoyl)pyridin-2-yl)cyclopropanecarboxamide(0.50 g, 1.2 mmol) was suspended in 25 mL of methanol. Sodiumborohydride (0.454 g, 12.0 mmol) was added in small portions. Afterstirring for one hour the reaction mixture was evaporated to dryness andpurified on 40 g of silica gel utilizing a gradient of 15-70% ethylacetate in hexanes to yield the pure product as a white solid (0.2125 g,0.5078 mmol, 42%). ESI-MS m/z calc. 418.6, found 419.3 (M+1)+. Retentiontime 2.59 minutes.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide(41.8 mg, 0.100 mmol) was suspended in 2 mL of toluene containing4-methylbenzenesulfonic acid hydrate (pTsOH, 23.8 mg, 0.125 mmol) andpropane-1,3-diol (9.89 mg, 0.130 mmol). The reaction mixture was thenheated to 140° C. for 5 minutes in a microwave reactor. The crudeproduct was then filtered, evaporated to dryness, dissolved inN,N-dimethylformamide (1 mL) and purified by reverse-phase preparativeliquid chromatography utilizing a gradient of 0-99% acetonitrile inwater containing 0.05% trifluoroacetic acid to yield the pure product(26.5 mg, 0.0556 mmol, 55.6%). ESI-MS m/z calc. 476.5, found 477.3(M+1)⁺. Retention time 2.71 minutes.

Preparation of1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(dimethylamino)ethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(dimethylamino)ethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand 2-(dimethylamino)ethanol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(2-(piperidin-1-yl)ethoxy)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(2-(piperidin-1-yl)ethoxy)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand 2-(piperidin-1-yl)ethanol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(propoxy)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(propoxy)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand propan-1-ol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl(pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(diisopropylamino)ethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(diisopropylamino)ethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand 2-(diisopropylamino)ethanol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(2-morpholinoethoxy)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(2-morpholinoethoxy)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand 2-morpholinoethanol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-hydroxyethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-hydroxyethoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(hydroxy(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamideand ethane-1,2-diol in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((3-hydroxypropoxy)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

Step a:(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate

Methanesulfonyl chloride (56 μl, 0.72 mmol) was added slowly to themixture of 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[hydroxy-(2-methoxy-phenyl)-methyl]-pyridin-2-yl}-amide (50 mg, 0.12mmol) and DIEA (24 μl, 0.14 mmol) in dichloromethane (2 ml) at 0° C. Thereaction mixture was stirred at ambient temperature for 3 h. Solventswere evaporated and the residue was used for the next step without anyadditional purification.

Step b:1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

(6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate (0.12 mmol) was dissolved in NMP (2 ml) following byaddition of 2M solution of dimethylamine in THF (600 μl, 0.12 mmol) andDIEA (21 μl, 0.12 mmol). The reaction mixture was maintained at ambienttemperature for 16 h. The resulting mixture was subjected to HPLCpurification to provide the trifluoroacetic salt of target material (55mg, 82%) as a colorless solid. ¹H-NMR (DMSO): δ 8.43 (s, 1H), δ 8.40 (d,J=2 Hz, 1H), δ 8.06 (d, J=8.7 Hz, 1H), δ 7.96 (dd, J=2 Hz, J=8.7 Hz,1H), δ 7.56 (bd, J=6.5 Hz, 1H), δ 7.39 (bt, J=6.5 Hz, 1H), δ 7.05-7.11(m, 3H), δ 6.96 (s, 2H), δ 6.05 (s, 2H), δ 5.68 (bm, 1H), δ 3.85 (s,3H), δ 2.75 (s, 6H), δ 1.42-1.45 (m, 2H), δ 1.13-1.16 (m, 2H).

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(methylamino)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(methylamino)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and methyl amine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((ethyl(methyl)amino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((ethyl(methyl)amino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and N-methylethanamine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(pyrrolidin-1-yl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(pyrrolidin-1-yl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and pyrrolidine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]-dioxol-5-yl)-N-(5-((diethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((diethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and diethylamine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(piperidin-1-yl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(piperidin-1-yl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and piperidine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(((R)-3-hydroxypyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(((R)-3-hydroxypyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and (R)-3-hydroxypyrrolidine in a manner analogous tothat of1-(benzo[d][1,3]dioxo-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(morpholino)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-methoxyphenyl)(morpholino)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and morpholine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((4-hydroxypiperidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((4-hydroxypiperidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and 4-hydroxypiperidine in a manner analogous to thatof1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-((6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methyl)pyrrolidine-2-carboxamide

1-((6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methyl)pyrrolidine-2-carboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and pyrrolidine-2-carboxamide in a manner analogous tothat of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-(((S)-2-(methoxymethyl)pyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(((S)-2-(methoxymethyl)pyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and piperidine in a manner analogous to that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2,6-dimethylmorpholino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2,6-dimethylmorpholino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and 2,6-dimethylmorpholine in a manner analogous tothat of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(methoxymethyl)pyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((2-(methoxymethyl)pyrrolidin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and 2-(methoxymethyl)pyrrolidine in a manner analogousto that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((4-(2-hydroxyethyl)piperazin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((4-(2-hydroxyethyl)piperazin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)methylmethanesulfonate and 2-(piperazin-1-yl)ethanol in a manner analogous tothat of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Preparation of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((4-(2-methoxyethyl)piperazin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide

1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-((4-(2-hydroxyethyl)piperazin-1-yl)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamidewas prepared from(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-3-yl)(2-methoxyphenyl)ethylmethanesulfonate and 1-(2-methoxyethyl)piperazine in a manner analogousto that of1-(benzo[d][1,3]dioxol-5-yl)-N-(5-((dimethylamino)(2-methoxyphenyl)methyl)pyridin-2-yl)cyclopropanecarboxamide.

Set forth below is the characterizing data for compounds of the presentinvention prepared according to the above examples.

TABLE 5 Cmpd. LC/MS LC/RT No. M + 1 min. 1 403.1 3.48 2 311.1 2.26 3403.1 3.24 4 443.5 3.4 5 387.1 3.44 6 326.3 2.33 7 474.3 2.94 8 389.33.2 9 472.3 2.88 10 419.3 2.91 11 532.3 2.39 12 433.3 3.3 13 389.1 2.8914 387.1 3.45 15 390.3 2.93 16 379.3 8.16 17 351.1 3.5 18 546.5 2.59 19297.1 2.67 20 446.3 2.8 21 297.3 2.09 22 545.5 2.74 23 516.3 2.96 24441.3 8.81 25 269.1 2.82 26 311.3 2.4 27 417.3 3.5 28 403.5 3.57 29339.3 6.6 30 457.5 3.79 31 373.3 3.33 32 486.3 2.94 33 461.5 3.6 34389.1 2.97 35 403.3 3.15 36 516.3 2.97 37 419.3 3.69 38 283.1 2.68 39531.5 2.61 40 333.1 1.48 41 345.1 3.07 42 297.3 2.18 43 325.3 2.13 44419.3 2.57 45 515.5 2.75 46 393.1 3.3 47 301.3 3.05 48 389.1 2.55 49488.3 2.8 50 453 1.63 51 516.3 3.01 52 404.5 2.73 53 432.3 2.74 54 283.12.61 55 488.3 2.76 56 339.2 3.19 57 463.5 2.66 58 502.3 2.75 59 407.53.39 60 403.5 3.11 61 267.1 1.6 62 460.3 2.86 63 373.1 7.14 64 404.53.01 65 308.1 3.07 66 477.3 2.71 67 379.1 3.56 68 453.1 3.69 69 283.12.13 70 387.3 3.7 71 530.5 2.53 72 297.3 2.19 73 438.3 1.83 74 418.32.87 75 297.1 2.48 76 407.5 3.69 77 373.1 3.5 78 407 3.6

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds.

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. Assay Plates containing cells are incubated for ˜2-4 hours intissue culture incubator at 37° C., 5% CO₂, 90% humidity. Cells are thenready for compound exposure after adhering to the bottom of the assayplates.

The cells were incubated in serum-free medium for 16-24 hrs in tissueculture incubator at 37° C., 5% CO₂, 90% humidity in the presence orabsence (negative control) of test compound. The cells were subsequentlyrinsed 3× with Krebs Ringers solution and loaded with a voltage sensingredistribution dye. To activate ΔF508-CFTR, 10 μM forskolin and the CFTRpotentiator, genistein (20 μM), were added along with Cl⁻-free medium toeach well. The addition of Cl⁻-free medium promoted Cl⁻ efflux inresponse to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using voltage sensor dyes.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluoresecent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions:

Bath Solution #1: (in mM)NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds.

1. Ussing Chamber Assay

Ussing chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that wereprecoated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUssing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,Iowa). Briefly, HBE were examined under voltage-clamp recordingconditions (V_(hold)=0 mV) at 37° C. The basolateral solution contained(in mM) 145 NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apicalsolution contained (in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM), PDE inhibitor, IBMX (100 μM) and CFTR potentiator,genistein (50 μM) were added to the apical side.

As observed in other cell types, incubation at low temperatures of FRTcells and human bronchial epithelial cells isolated from diseased CFpatients (CF-HBE) expressing ΔF508-CFTR increases the functional densityof CFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with test compound for 24-48 hoursat 37° C. and were subsequently washed 3× prior to recording. The cAMP-and genistein-mediated I_(SC) in compound-treated cells was normalizedto 37° C. controls and expressed as percentage activity of CFTR activityin wt-HBE. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

2. Patch-Clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mlamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture,

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, 13-ME, I X pen/strep, and25 mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=l/i(N), where l=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

The compounds of Table 1 were found to exhibit Correction activity asmeasured in the assay described above.

Compounds of the invention are useful as modulators of ATP bindingcassette transporters. Using the procedures described above, theactivities, i.e., EC50s, of compounds of the present invention have beenmeasured and are shown in Table 6.

TABLE 6 IC50/EC50 Bins: +++ <= 2.0 < ++ <= 5.0 < + PercentActivityBins: + <= 25.0 < ++ <= 100.0 < +++ Cmpd. No. BinnedEC50BinnedMaxEfficacy 1 + ++ 2 + +++ 3 +++ +++ 4 + +++ 5 +++ +++ 6 + ++ 7+++ +++ 8 ++ ++ 9 ++ +++ 10 ++ ++ 11 ++ +++ 12 + ++ 13 ++ ++ 14 ++ +++15 ++ ++ 16 +++ +++ 17 ++ ++ 18 +++ ++ 19 + ++ 20 ++ +++ 21 + +++ 22 +++++ 23 +++ +++ 24 +++ +++ 25 + ++ 26 ++ +++ 27 ++ +++ 28 + +++ 29 ++ +++30 + ++ 31 ++ ++ 32 +++ +++ 33 ++ +++ 34 ++ ++ 35 +++ +++ 36 +++ +++ 37++ +++ 38 + ++ 39 +++ ++ 40 ++ +++ 41 + ++ 42 + +++ 43 + ++ 44 + +++ 45++ +++ 46 ++ +++ 47 + +++ 48 + ++ 49 +++ +++ 50 ++ ++ 51 +++ +++ 52 ++++++ 53 ++ +++ 54 + ++ 55 ++ +++ 56 + +++ 57 ++ +++ 58 ++ +++ 59 +++ +++60 +++ +++ 61 + ++ 62 ++ +++ 63 +++ +++ 64 +++ ++ 65 + ++ 66 ++ +++ 67++ +++ 68 ++ +++ 69 + +++ 70 ++ +++ 71 +++ ++ 72 + +++ 73 +++ +++ 74 ++++++ 75 + ++ 76 + +++ 77 + ++ 78 +++ +++

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R^(N) is H,alkyl, aryl, heteroaryl, aralkyl, cycloalkyl, or heterocycloalkyl; A isan optionally substuted 3-7 membered monocyclic ring; B is optionallyfused to a 5-7 membered ring selected from the group consisting ofcycloaliphatic, aryl, heterocyclic, and heteroaryl; R¹ is halo, alkyl,OH, alkoxy, thioalkyl, trifluoromethoxy; or two R¹ on adjacent atoms,taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;W is independently a bond or a (C1-C6) alkylidene chain wherein up totwo methylene units of W are independently replaced by —CO—, —O—, —S—,—SO₂—, or —NR′—; R′ is independently H, alkyl, aryl, heteroaryl,aralkyl, cycloalkyl, or heterocyclic; R^(W) is independently H, halo,CN, NO₂, N(R)₂, CF₃, OCF₃, OH, OR, C(O)R, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²; R² ishalo, CN, NO₂, CF₃, OCF₃, OR, OC(O)R, OC(O)N(R)₂, SR, S(O)R, SO₂R,SO₂N(R)₂, SO₃R, C(O)R, CO₂R, C(O)N(R)₂, N(R)₂, NRC(O)R, NRCO₂R,NRC(O)N(R)₂, NRSO₂R, B(OR)₂, or NRSO₂N(R)₂; R is independently H, alkyl,cycloalkyl, heterocyclic, aryl, or heteroaryl; n is 1 or 2; w is aninteger from 0 to 4 inclusive; and x is an integer from 0 to 5inclusive; provided that when W is a bond and R^(w) is cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, —W—R^(w) is attached to the3- or 4-position of the pyridyl ring.
 2. The compound of claim 1,wherein A is selected from the group consisting of:

wherein R³ is alkyl, alkaryl, aryl, or heteroaryl; and q is an integerfrom 0 to 4 inclusive.
 3. The compound of claim 2, wherein A is


4. The compound of claim 2, wherein A is


5. The compound of claim 1, wherein R¹ is selected from the groupconsisting of chloro, methyl, OH, methoxy, thiomethyl, andtrifluoromethoxy.
 6. The compound of claim 1, wherein two R¹ on adjacentatoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂.7. The compound of claim 6, wherein J is CH₂.
 8. The compound of claim6, wherein J is CF₂.
 9. The compound of claim 1, wherein R^(N) is H oralkyl.
 10. The compound of claim 1, wherein W is a bond.
 11. Thecompound of claim 1, wherein W is an optionally substituted (C1-C6)alkylidene chain.
 12. The compound of claim 1, wherein W is selectedfrom the group consisting of —CH₂—, —NH—, —O—, —CO—, and —OCH₂—.
 13. Thecompound of claim 1, wherein R^(w) is selected from the group consistingof H, OH, heterocyclic, aryl, phenyl, heteroaryl, pyridyl, alkoxy,cycloalkyl, and cyclohexyl.
 14. The compound of claim 1, wherein—W—R^(w) is acyclic.
 15. The compound of claim 1, wherein —W—R^(w) is anaryl, heteroaryl, cycloaliphatic, or heterocycloaliphatic ring.
 16. Thecompound of claim 1, wherein —W(R^(w))_(n) is selected from thefollowing: —F, —CH₃, —CH₂CH₃, —CN, —CF₃, —CONH₂, —CH₂CH(CH₃)₂,


17. The compound of claim 1, wherein —W(R^(w))_(n) selected from thefollowing:


18. The compound according to claim 1, having formula Ia:

or a pharmaceutically acceptable salt thereof, wherein: J is CH₂ or CF₂;W is independently a bond or a (C1-C6) alkylidene chain optionallysubstituted with R² wherein up to two methylene units of W areindependently replaced by —CO—, —O—, or —NR′—; R′ is independently H oralkyl; R^(W) is independently H, halo, CN, N(R)₂, CF₃, OH, CO₂R,C(O)N(R)₂, —O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²; R² ishalo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂; R isindependently H, alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl; nis 1 or 2; and w is an integer from 0 to 4 inclusive; provided that whenW is a bond and R^(w) is cycloaliphatic, heterocycloaliphatic, aryl, orheteroaryl, —W—R^(w) is attached to the 3- or 4-position of the pyridylring.
 19. The compound of claim 18, wherein J is CH₂.
 20. The compoundof claim 18, wherein J is CF₂.
 21. The compound of claim 18, wherein—W—R^(w) is acyclic.
 22. The compound of claim 18, wherein —W—R^(w) isan aryl, heteroaryl, cycloaliphatic, or heterocycloaliphatic ring. 23.The compound of claim 18, wherein —W(R^(w))_(n) is selected from thefollowing: —F, —CH₃, —CH₂CH₃, —CN, —CF₃, —CONH₂, —CH₂CH(CH₃)₂,


24. The compound of claim 18, wherein —W(R^(w))_(n) selected from thefollowing:


25. The compound according to claim 1, having formula Ib:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is halo,alkyl, OH, alkoxy, thioalkyl, trifluoromethoxy; or two R¹ on adjacentatoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;W is —NR′, —CO—, or —CH₂—; R′ is independently H or alkyl; R^(W) isindependently H, halo, CN, N(R)₂, CF₃, OH, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²; R² ishalo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂; R isindependently H, alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl; nis 1 or 2; w is an integer from 0 to 4 inclusive; and x is an integerfrom 0 to 5 inclusive.
 26. The compound of claim 25, wherein two R¹ onadjacent atoms, taken together, form

and J is CH₂.
 27. The compound of claim 25, wherein two R¹ on adjacentatoms, taken together, form

and J is CF₂.
 28. The compound of claim 25, wherein —W(R^(w))_(n) isselected from the following: —CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂,


29. The compound according to claim 1 having formula Ic:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is halo,alkyl, OH, alkoxy, thioalkyl, trifluoromethoxy; or two R¹ on adjacentatoms, taken together, form

wherein J is selected from the group consisting of CH₂, CF₂, or C(CH₃)₂;W is —NR′, —CO—, or —CH₂—; R′ is independently H or alkyl; R^(W) isindependently H, halo, CN, N(R)₂, CF₃, OH, CO₂R, C(O)N(R)₂,—O(C1-C6)alkylidene-OR, —O(C1-C6)alkylidene-N(R)₂,—O(C1-C6)alkylidene-heterocyclic, or an optionally substitutedaliphatic, cycloaliphatic, aryl, aryloxy, heterocyclic, or heteroaryl,wherein, when substituted, R^(w) is substituted with up to two R²; R² ishalo, OR, CO₂R, C(O)N(R)₂, SO₂N(R)₂, B(OR)₂, or N(R)₂; R isindependently H, alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl; nis 1 or 2; w is an integer from 0 to 4 inclusive; and x is an integerfrom 0 to 5 inclusive.
 30. The compound of claim 29, wherein two R¹ onadjacent atoms, taken together, form

and J is CH₂.
 31. The compound of claim 29, wherein two R¹ on adjacentatoms, taken together, form

and J is CF₂.
 32. The compound of claim 29, wherein —W(R^(w))_(n) isselected from the following: —CH₃, —CH₂CH₃, —CH₂CH(CH₃)₂,


33. The compound of claim 1, wherein the compound is selected fromTable
 1. 34. A pharmaceutical composition comprising (i) a compound ofclaim 1; and (ii) a pharmaceutically acceptable carrier.
 35. Thecomposition of claim 34, further comprising an additional agent selectedfrom the group consisting of a mucolytic agent, bronchodialator, ananti-biotic, an anti-infective agent, an anti-inflammatory agent, CFTRcorrector, and a nutritional agent. 36-40. (canceled)
 41. A kit for usein measuring the activity of a ABC transporter or a fragment thereof ina biological sample in vitro or in vivo, comprising: (i) a firstcomposition comprising a compound according to claim 1; and (ii)instructions for: a) contacting the composition with the biologicalsample; b) measuring activity of said ABC transporter or a fragmentthereof.